Molecular Vision 2006; 12:26-31 <http://www.molvis.org/molvis/v12/a3/>
Received 27 September 2005 | Accepted 4 January 2006 | Published 10 January 2006
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A missense mutation in the γD-crystallin gene GRYGD associated with autosomal dominant congenital cataract in a Chinese family

Feng Gu,1,2 Rong Li,3 Xi Xin Ma,3 Li Song Shi,2,4 Shang Zhi Huang,2,4 Xu Ma1,2,5
 
 

1Department of Genetics, National Research Institute for Family Planning, Beijing, China; 2Peking Union Medical College, Beijing, China; 3Zhengzhou Er Qi District Hospital, Zhengzhou, Henan, China; 4Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China; 5Department of Reproductive Genetics, WHO Collaborative Center for Research in Human Reproduction, Beijing, China

Correspondence to: Xu Ma, National Research Institute for Family Planning, 12 Da-hui-si, Hai Dian, Beijing, 100081, China; Phone: +86-010-62176870; FAX: +86-010-62179059; email: genetic@263.net.cn


Abstract

Purpose: To identify the genetic defect in autosomal dominant congenital cataracts in a six generation Chinese family.

Methods: Clinical and ophthalmological examinations were performed on the affected and unaffected family members. All the members were genotyped with microsatellite markers at loci which were considered to be associated with cataracts. A two-point LOD score was calculated using the Linkage package after genotyping. A mutation was detected by direct sequencing using gene specific primers.

Results: Clinical heterogeneity was observed within this family, three affected individuals showed nuclear cataract and others had coralliform cataracts. Significant evidence of linkage was obtained at markers D2S325 (LOD score [Z]=3.10, recombination fraction [θ]=0.0) and D2S1782 (Z=5.97, θ=0.0), respectively. Haplotype analysis indicated that the cataract gene was close to those two markers. Sequencing of the γD-crystallin gene (CRYGD) revealed a C>T transition in exon 2, that causes a conservative substitution of Arg to Cys at codon 14 (R14C). This mutation co-segregated with all affected individuals and was not observed in unaffected or 100 normal unrelated individuals. Bioinformatic analyses also showed that a highly conserved region was located at Arg14.

Conclusions: This study is the first reported case with phenotype of coralliform/nuclear cataract that associated with the mutation of Arg14Cys (R14C) CRYGD.


Introduction

Hereditary cataract is a clinically and genetically heterogeneous lens disease that induced a significant proportion of visual impairment and blindness in childhood [1,2]. Over the past few years, mutations in several functionally diverse genes have now been identified in a small percentage of congenital cataract patients, which included: CRYAA [3], CRYAB [4], CRYBA1/A3 [5], CRYBB1 [6], CRYBB2 [7,8], CRYGC [9,10], CRYGD [11], GJA3 [12], GJA8 [13,14], MIP [15], BFSP2 [16,17], PITX3 [18], HSF4 [19], and LIM2 [20].

Even though the hereditary aspects of congenital cataracts have been recognized for nearly a century and some progress has been made, the relationship between morphology and genetic etiology of congenital cataract is not clear yet, this is due to the complex developmental process of the ocular lens and a wide variation in opacity morphology.

According to the specific morphology of the lens concerned with position and appearance, under slit lamp examination, congenital cataracts were classified as anterior polar, posterior polar, nuclear, lamellar (zonular), pulverulent, aculeiform, cerulean, cortical, polymorphic, sutural, coralliform, and total cataract [21].

In this study, we detected linkage of an autosomal dominant inherited coralliform/nuclear cataract with the D2S325 and D2S1782 markers in a six generation Chinese family. A missense mutation (C34T) in CRYGD was identified, which resulted in an Arg to Cys (R14C) substitution in CRYGD. To our knowledge, this is the first reported case with phenotype of coralliform/nuclear cataracts caused by C34T mutation in CRYGD.


Methods

Clinical evaluations and DNA specimens

The protocol for the study was approved by the Ethics Committee of our institute. Informed consent was obtained from all family members participating in this study. The family comprised 25 affected individuals from a six generation pedigree (Figure 1), originating from the province of Henan, China. The study consisted of 30 members, including 16 affected individuals, 7 unaffected individuals, and 7 spouses (Figure 1). Clinical and ophthalmological examinations were performed. The diagnosis of cataract was confirmed by ophthalmologists. There was no history of other ocular or systemic abnormalities in the family.

Genotyping

Exclusion analysis was performed by allele sharing of the microsatellite markers, which were linked with known cataract loci, on affected individuals. Genotyping was performed as described previously [22] using microsatellite markers D2S325 and D2S1782 at 2q33-35, which are linked with the coralliform cataract. The oligonucleotide primer sequences were taken from NCBI and GDB.

Linkage analysis

A two-point LOD score (Z) was calculated using the MLINK subprogram of the Linkage package (version 5.1). The mode of inheritance was considered to be autosomal dominant with full penetrance. The gene frequency was set at 1/10,000. As the allelic frequencies of the polymorphic markers are unknown in the Chinese population, they were considered to be equally distributed.

DNA sequencing

Mutations in CRYGA, CRYGB, CRYGC, and CRYGD were screened by direct sequencing. PCR products of the three exons and flanking intron sequences of CRYGA, CRYGB, CRYGC, and CRYGD [23] were sequenced on an ABI A377 Automated Sequencer (PE Biosystems, Foster City, CA).

Denaturing HPLC

Denaturing (D)HPLC was used to screen the mutation identified in the patients, on remaining patients, family members, and 100 normal control subjects in exon 2 of the CRYGD gene using a commercial system (Wave DHPLC; Transgenomic, San Jose, CA). Gene specific PCR primers were used to amplify the fragment which harbored the point mutation. DHPLC was performed as follows: initial concentration at 48% of buffer A (0.1 M triethylammonium acetate-TEAA; Transgenomic), and 52% of buffer B (0.1 M TEAA containing 25% acetonitrile; Transgenomic) at 65 °C.


Results

Clinical data

The proband was a 17-year-old male (IV:9) who had a bilateral cataract in 1993. The form of the opacification was irregular as sea coral, with crystal clumps radiating from center to capsule. Except for affected individuals IV:1, IV:2, and V:16 with nuclear opacification, all the affected individuals showed a phenotype of coralliform cataract (age of VI:1 was 5 and IV:7 was 38; Figure 2A,B). Slit-lamp photographs of the affected individuals (IV:1, IV:5, V:16; the age of IV:1 was 54; Figure 2C) showed that opacities were located mainly in the nuclear areas of both lenses and there were pulverulent opacities in the perinuclear areas, which are different from the coralliform cataract. Several of the affected individuals had high myopia with elongated axis oculi; ocular lengths were (OD/OS) 29.98 mm/30.54 mm, 27.58 mm/28.61 mm, 29.02 mm/29.66 mm, 24.1 mm/24.5 mm in individuals IV:1, IV:5, V:4, and VI:1, respectively. No systemic or other ocular anomalies were observed in the patients.

Linkage and haplotype analysis

Allele-sharing analysis excluded the linkage of the disease in the family with all known loci of cataract except that for CRYGs, D2S325, and D2S1782 at 2q33 (data not shown). Haplotype analysis showed that the affected individuals in the family shared a common haplotype with markers D2S325 and D2S1782 at 2q33 (Figure 1). Significant evidence of linkage was observed with microsatellite markers D2S325 (LOD score [Z]=3.10, at recombination fraction [θ]=0.0) and D2S1782 (Z=5.97, at θ=0.0), respectively (Table 1). This implied that one of the members of CRYG in the region might be responsible for the disease.

Mutation detection for CRYGA, CRYGB, CRYGC, and CRYGD

Direct cycle sequencing of the amplified fragments of CRYGs in two affected individuals identified a single base alteration C34T (Figure 3A) in exon 2 of CRYGD (NM_006891), which resulted in a substitution of Arg to Cys at codon 14 (R14C). Denaturing HPLC analysis confirmed this mutation, which co-segregated with all affected individuals in the family, and were not observed in any of the unaffected family members or 100 normal controls. The remainder of the coding sequence did not show any sequence change.

Multiple-sequence alignment and mutation analysis

Using the NCBI and UCSC websites, we obtained multiple-sequence alignment of the CRYG family proteins in various species with DNAMAN biosoftware, including Homo sapiens, Canis familiaris, Mus musculus, and Rattus norvegicus (Figure 3B). We found that codon 14, where mutation (R14C) occurred, located within a highly conserved region.

Furthermore, we used online bioinformatics software SIFT [24] to predict whether the amino acid substitution in CRYGD could have a phenotypic effect, with a result of the substitution at position 14 from R to C is predicted to affect protein function with a score of 0.00. While positions with normalized probabilities less than 0.05 are predicted to be deleterious, those greater than or equal to 0.05 are predicted to be tolerated. All of these indicated that the Arg-14 is an important residue for the function of CRYGD.


Discussion

Coralliform cataract is an uncommon form of congenital cataract, which was named for the morphological resemblance of these lens opacities to sea coral, extending from the nucleus into the anterior and posterior cortex. It was first reported in 1895 [25] and subsequently described as an autosomal dominant trait in three British pedigrees circa in 1910 [26]. Since then, this phenotype has been seldom reported. Phenotypes of aceuliform and fasciculiform cataract are also associated with needle-like projections extending from the nucleus into the anterior and posterior cortex, there may well be overlap among the three phenotypes. Some results [9,23,27,28] showed that the mutations of CRYGD, locating on 2q33-35, were responsible for coralliform, aceuliform, and fasciculiform phenotype, respectively. Recently, a new locus for coralliform cataract has been mapped to chromosome 2p24-pter in a four generation Chinese family [29]. There are, therefore, two genetic loci reported to date for coralliform cataract at 2q33-35 and 2p24-pter.

In this study, according to the phenotype of congenital cataracts, members of the family were firstly genotyped with microsatellite markers at all known loci of congenital cataracts, and special attention was paid to markers on 2q33-35 as there was allele sharing. Haplotype analysis showed that the affected individuals in this family shared a common haplotype with two markers, D2S325 and D2S1782, within the region of 2q33-35, where the members of the CRYG family locate.

The γ-crystallin gene cluster comprises six genes; CRYGA, CRYGB, CRYGC, CRYGD, CRYGE, and CRYGF. In mammals, each of these genes consists of three exons, only CRYGC and CRYGD encode abundant lens γ-crystallins in human [30,31]. CRYGE and CRYGF are pseudogenes with in-frame stop codons. CRYGC/D is one of the only two γ-crystallins to be expressed at high concentrations in the fiber cells of the embryonic human lens, which subsequently forms lens nucleus fibers. For this reason and with the phenotype, we focus our attention on CRYGD. After screening for mutations in CRYGA, CRYGB, CRYGC, and CRYGD by direct cycle sequencing, we identified a C>T transition in exon 2 of GRYGD, which presented only in affected members of the family. The transition C34T locates in exon 2, which was predicted to cause a conservative substitution of Arg to Cys at codon 14 (R14C). The results of multiple-sequence alignment and mutation analysis also confirmed the importance of Arg14 for the function of CRYGD.

Mutations in the γD-crystallin encoding gene (CRYGD) have been demonstrated to be one of the most frequent reasons for isolated congenital cataracts. Mutations within CRYGD so far reported were responsible for several phenotypes; progressive juvenile-onset punctate (R14C) [11], cerulean (P23T) [32], prismatic (R36S) [33], aceuliform (R58H) [9,27], nuclear (W156X) [34], lamellar (P23T) [34], fasciculiform (P23T) [28], and coralliform cataract (P23T) [23,27]. Functional studies on the mutation of CRYGD polypeptides in vitro also have shown that polypeptides with R14C, P23T, and R58H mutations are less soluble and more prone to crystallization [35-37]. All these results revealed that the CRYGD plays an important role in maintaining lens transparency.

Stephan et al. [11] have reported a C>T mutation at nucleotide 34 of CRYGD exon 2 in a family with juvenile-onset punctate cataract, which was identical to our study. Compared with affected individuals in that family (progressive trait), there was not enough evidence to prove that the progressive trait of the affected individuals in this Chinese family was not due to long-term follow-up. Meanwhile, there was no obvious progressive development in this Chinese family since the two affected individuals at different ages have similar lens opacities (Figure 2). The difference of phenotype cataract between the two families was significant; punctate cataract in Stephan's case, but coralliform/nuclear form in this report. The discrepancy is consistent with the fact that the relationship between morphology and genetic etiology of congenital cataract was not reliable in different populations [7,9,11,23,24,27,28,32-34,38].

In this family, there are two different phenotypes, coralliform and nuclear cataracts. Intrafamilial variation implied that clinically differentiation of both types of opacifications could be attributed to the action of other modifier genes.

High myopia is frequently seen in affected individuals, who have elongated ocular axis, suggesting that the mutation in this family may play a role in the development of high myopia. Further studies need to provide further insights into the molecular pathology of high myopia in this family.

Allele sharing analysis was a quick procedure for excluding a reported locus responsible for the disease in a family under study. If no allele sharing has been detected in affected individuals, there could be no linkage with the locus. Otherwise, a further linkage analysis was required for confirmation since shared alleles could come from the unaffected parent. Under linkage analysis a higher LOD score has been obtained at marker D2S1782 (Z=5.97) but not at D2S325 (3.10) in this family, while D2S325 lies more closely to the disease gene (Figure 1). The reason for it was that more individuals were homozygous for D2S325 in the family than that for D2S1782.

This study is the first example of Intrafamilial variability in the cataract phenotype associated with R14C CRYGD mutation. The clinical variability within the family or between the two families can be attributed to the action of other modifier genes or, perhaps less likely, environmental factors. In summary, our results provided evidence for clinical heterogeneity of punctate, coralliform, and nuclear cataracts. Our results further confirmed that CRYGD is important in the maintenance of optical clarity.


Acknowledgements

The authors thank the family for their participation in this project and Dr. Siquan Zhu (Beijing Tongren Hospital, Capital University of Medical Sciences, Beijing), Dr. Guangying Zheng (the First affiliated Hospital, Zhengzhou University) for phenotype identification, Dr. Xuemin Jin for sample collection, and Dr. Xiaohui Yang for photographic records. This work is partly supported by the National "973" Basic Research Funding Scheme of China (grant number 2001CB5103) and National Infrastructure Program of Chinese Genetic Resources (2004DKA30490).


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